Screening method for modulation of human mast cell activation

ABSTRACT

Human mast cell activation is modulated by ATP binding to P2-purinoceptors on the mast cell surface. ATP binding to the purinoceptors provides a target for therapeutic intervention for the treatment of disorders characterized by undesireable mast cell mediator release, such as asthma and allergy. Inhibitors of ATP binding to mast cell P2-purinoceptors are useful therapeutic agents for treatments of those disorders. Methods of treatment using agents, and in vitro screening assays for selection of the therapeutic agents, are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.09/381,692, filed Dec. 2, 1999, patented U.S. Pat. No. 6,372,724 B1,which is a 371 of PCT/US98/05922, filed Mar. 24, 1998, abandoned, whichclaimed priority from U.S. provisional patent application No.60/041,461, filed Mar. 25, 1997, abandoned.

REFERENCE TO GOVERNMENT GRANT

The invention was supported in part by grant AI 20634 from the NationalInstitutes of Health. The United States government has certain rights inthe invention.

FIELD OF THE INVENTION

The invention relates to the modulation of human mast cell activation bycompounds which modulate adenosine 5′-triphosphate (ATP) binding to ATPreceptors (P2-purinoceptors) on the cells. The invention further relatesto the treatment of disorders characterized by undesirable mediatorrelease from stimulated mast cells, particularly immunologicallystimulated lung mast cells. The invention also relates to methods for invitro screening of candidate therapeutic agents for treating suchdisorders.

BACKGROUND OF THE INVENTION

Mast Cells

Mast cells comprise a normal component of the connective tissue thatplays an important role in immediate (type I) hypersensitivity andinflammatory reactions by secreting a large variety of chemicalmediators from storage sites in their granules upon stimulation. Mastcells, and their circulating counterparts the basophils, possess surfacereceptors known as FcεRI. The receptors are specific for antibody εheavy chains.

The event that initiates immediate hypersensitivity is the binding ofantigen to IgE on the mast cell or basophil surface. Both cell types areactivated by cross-linking of FcεRI molecules, which is thought to occurby binding multivalent antigens to the attached IgE molecules.

Mast cells may also be activated by mechanisms other than cross-linkingFcεRI, such as in response to mononuclear phagocyte-derivedchemocytokines, to T cell-derived cytokines and to complement-derivedanaphylatoxins. Mast cells may also be recruited and activated by otherinflammatory cells or by neurotransmitters which serves as links to thenervous system.

When antigen binds to IgE molecules attached to the surface of mastcells, a variety of mediators are released which give rise to increasedvascular permeation, vasodilation, bronchial and visceral smooth musclecontraction, and local inflammation. In the most extreme form ofimmediate hypersensitivity reaction known as anaphylaxis, mediatorsreleased from mast cells can restrict airways to the point ofasphyxiation. So-called atopic individuals, who are prone to developstrong immediate hypersensitivity responses, may suffer from asthma, hayfever or chronic eczema. These individuals possess higher than averageplasma IgE levels.

Antigens that elicit strong immediate hypersensitivity reactions areknown as allergens. Allergy afflicts twenty percent of the United Statespopulation.

Immediate hypersensitivity results from the following sequence ofevents: production of IgE by B cells in response to antigen, binding ofthe IgE to FcεRI on the surface of mast cells, interaction ofre-introduced antigen with the bound IgE and activation of the mastcells and release of mediators. Antigen binding can be simulated bypolyvalent anti-IgE or by anti-FcεRI antibodies. Such antibodies canactivate mast cells from atopic as well as non-atopic individuals,whereas allergens activate mast cells only in atopic persons.

Mediators released from mast cells may be divided into two broadclasses, pre-formed or secretory granule associated mediators andnonpreformed or newly synthesized mediators. The pre-formed mediatorsinclude biogenic amines, most notably histamine. The pre-formedmediators also comprise granule macromolecules such as proteoglycans,most notably heparin and chondroitin sulfate E; chemotactic factors suchas eosinophil and neutrophil chemotactic factors of anaphylaxis; andenzymes such as proteases, tryptase, chymase, cathepsin G-like enzyme,elastase, carboxypeptidase A and acid hydrolases. The nonpreformedmediators include products of arachidonic acid, prostaglandin D₂,leukotrienes C₄ and B₄ and platelet activating factor. Another class ofmediators, the cytokines, are produced by mast cells upon IgE-mediatedactivation, or by other cells, including recruited T_(H)2 lymphocytes.The cytokines are predominantly responsible for the late phase reactionwhich begins two to four hours after elicitation of many immediatehypersensitivity reactions. One cytokine, tumor necrosis factor alpha,may exist in the mast cells as preformed stores, or may represent anewly synthesized product released over a period of hours.

Mediators released from human mast cells are central to thepathophysiology of allergy, asthma and anaphylaxis. In particular, mastcells and their release of histamine and other mediators play animportant role in the symptomatology of asthma and other human diseases.During the early phase of human lung hypersensitivity reactions uponexposure to antigen (i.e., pollens, cats, etc.), mast cells release andare the major source of histamine, and newly synthesized lipid productsof arachidonic acid metabolism: prostaglandin D₂ and leukotriene C₄.These mediators produce immediate breathlessness, which subsides in onehour but returns within 2-4 hours (the “late phase” response). Attestingto their primal role in hypersensitivity responses, human lung mastcells (HLMC) are characterized by mRNA generation, protein synthesis andrelease of so-called T_(H)2 cytokines within these first few hours ofactivation. These cytokines including IL-5, and IL-13 are believed to becentral to the evolution of chronic allergic/asthmatic states. In thelung, only mast cells are a source of histamine. Thus, histamine releaseis a distinct marker of mast cell activation and behavior. For a reviewof the role of mast cells in inflammatory responses in the lung, seeSchulman, Critical Reviews in Immunology, 13(1):35-70 (1993), the entiredisclosure of which is incorporated herein by reference.

Clinically, asthma is recognized by airway hyperactivity and reversibleairways obstruction. Pathological derangements at the tissue levelinclude constriction of airway smooth muscle, increased vascularpermeability resulting in edema of airways, outpouring of mucus fromgoblet cells and mucus glands, parasympathetic nervous systemactivation, denudation of airway epithelial lining cells, and influx ofinflammatory cells. Underlying these tissue effects are direct effectsof potent mediators secreted following physical, inflammatory, orimmunological activation and degranulation. The early phase of theasthmatic reaction is mediated by histamine and other mast cellmediators that induce rapid effects on target organs, particularlysmooth muscle. The pathophysiologic sequence of asthma may be initiatedby mast cell activation in response to allergen binding to IgE. Evidenceexists to link exercise-induced asthma and so-called “aspirin-sensitive”asthma to HLMC degranulation.

Pharmacologic Modulation of Mast Cell Function

A limited number of pharmacologic agents have been tested for effect onHLMC activation-secretion. The beta-adrenergic agonist pharmacologicagents, as typified by fenoterol, are the most potent global inhibitorsof HLMC. Though widely touted as “mast cell stabilizers,” disodiumcromoglycate and nedocromil sodium poorly inhibit purified HLMChistamine release. While certain corticosteroids have been found tosuppress IgE-mediated generation of late-phase cytokine mRNA and protein(e.g., IL-5), release of early phase mediators (e.g., histamine andLTC₄) are unaffected by corticosteroids. HLMC release has been shown tobe inhibited by the immunosuppressant agents FK-506, cyclosporin A andauranofin. Arachidonate pathway inhibitors are of considerableimportance, they may leave the release of other allergic mediators(e.g., histamine, proteases) unaffected. Such arachidonate pathwayinhibitors include inhibitors of 5-lipoxygenase and inhibitors ofcyclooxygenase.

Adenosine and Adenosine Triphosphate

ATP is found in every cell of the human body; it plays a major role incellular metabolism and energetics. ATP is released into theextracellular fluid under physiologic and pathophysiologic conditions.For example, ATP is released from ischemic cells, activated platelets,apoptotic and necrotic cells, nerve terminals as a co-transmitter, andmuscle fibers during exercise. Inhalation of aerosolized ATP has beenshown to trigger bronchoconstriction in healthy and asthmatic humansubjects (Pellegrino et al., J. Appl. Physiol. 81, 964-975, 1996). Onceoutside cells, ATP exerts different actions in various tissues andorgans. These actions are mediated by distinct cell surface receptors,termed P2-purinoceptors. These receptors are different from theadenosine receptors, termed P1-purinoceptors. This distinction ofdifferent receptors is critical, as adenosine is a breakdown product ofATP. The P2-purinoceptors comprise two major families, P2X and P2Y. Eachfamily consists of at least seven members (X₁₋₇ and Y₁₋₇). The P2Xfamily represents cell membrane ligand-binding ion channels permeable toNa⁺, K⁺, and Ca²⁺. The P2Y-purinoceptors constitute G-protein-linkedreceptors, often coupled to phospholipase C and, hence, to inositoltriphosphate formation. There are at least seven different subclasses ofP2Y receptor, based upon agonist potency profiles. For a description ofthe various P2Y subtypes, see Abbrachio and Burnstock, Pharmac. Ther.64, 445-475, 1994, the entire disclosure of which is incorporated hereinby reference.

ATP has been shown to induce histamine release from rat peritoneal mastcells (Keller, Tissue Mast Cells In Immune Reactions, S. Karger, p.38-39, 1966; Diamant, Int. Arch. Allergy 36:3-21, 1969; Sugiyama, Japan.J. Pharmacol. 21, 209-226, 1971; Cockcroft and Gomperts, J. Physiol 296,229-243, 1979). One study attempted to identify the receptor whichmediates the action of ATP on rat mast cells (Tatham et al., Euro. J.Pharmacol 147, 13-21, 1988). It was concluded in the study that thereceptor is actually stimulated by a minor component of ATP, termedATP⁴⁻ (Id.). ATP⁴⁻ effects are mediated through activation of theP2X₇-purinoceptor (previously termed the P2Z-purinoceptor) expressed onthe rat mast cell surface (Bennett et al., J. Physiol. (Lond.)317:335-345, 1981).

While rat studies suggest that ATP can directly induce mediator releasefrom lung mast cells, these results cannot necessarily be applied tohuman mast cells, as will be apparent from the following disclosure.

SUMMARY OF THE INVENTION

A method for inhibiting mediator release from stimulated human mastcells is provided. Human mast cells are contacted with an effectiveamount of an agent which inhibits ATP binding to P2-purinoceptors on thecells. Preferably, the agent inhibits ATP binding to a P2Y-purinoceptoron the cells, most preferably the P2Y₁- or P2Y₂-purinoceptor. The agentmay comprise, for example, a P2Y-purinoceptor antagonist or anallosteric modifier of a P2Y-purinoceptor.

According to one embodiment of the invention, the stimulated mast cellsso treated are mast cells which comprise immunologically stimulated mastcells. While the mast cells may be derived from any human tissue, theinvention is most advantageously practiced on lung, gut or joint mastcells.

According to another embodiment, the invention is a method for treatinga human subject for a disorder characterized by undesirable release ofmediator from immunologically stimulated lung mast cells. An effectiveamount of an agent which inhibits ATP binding to P2-purinoceptors onmast cells is administered to the subject. The disorder may, forexample, be a disorder characterized by the undesirable release ofhistamine, such as allergy or asthma. The disorder may also compriseinflammatory lung disease, or bronchoconstriction, such asbronchoconstriction associated with pulmonary embolism.

According to one particularly preferred embodiment of the invention, ahuman subject is treated for a bronchoconstriction caused by histaminerelease from stimulated lung mast cells by administration of aneffective amount of an agent which inhibits ATP binding to aP2-purinoceptor, preferably to a P2Y-purinoceptor, most preferably theP2Y₁- or P2Y₂-purinoceptor, on lung mast cells.

The invention also provides a method for selecting agents useful forinhibiting mediator release from stimulated human mast cells. The methodcomprises contacting stimulated human mast cells with an agent which isan inhibitor of ATP binding to a P2-purinoceptor, preferably aP2Y-purinoceptor, most preferably the P2Y₁- or P2Y₂-purinoceptor; andassaying said cells for release of one or more mediators. The stimulatedmast cells may comprise, for example, immunologically stimulated mastcells. Most preferably, the immunologically stimulated mast cellscomprise lung mast cells. The preferred mediator for assay is histamine.

The invention is also a method for determining, in vitro, theeffectiveness of an agent for the treatment of a human subject for adisorder characterized by undesirable release of mediator fromstimulated mast cells. The method is a competitive binding assay inwhich the test agent competes with a P2-purinoceptor ligand for bindingto a reagent comprising a P2-purinoceptor. The method comprises forminga mixture comprising the test agent, a P2-purinoceptor ligand(preferably a P2Y-purinoceptor ligand, most preferably a P2Y₁- orP2Y₂-purinoceptor ligand) and a reagent comprising a P2-purinoceptor(preferably a P2Y-purinoceptor, most preferably the P2y₁- orP2Y₂-purinoceptor); and assaying the mixture for the inhibition ofligand binding to the receptor by the agent. The ligand preferablycomprises a receptor agonist. The reagent may comprise, for example,human mast cells, particularly lung mast cells. The assay isparticularly useful for determining the effectiveness of agents for thetreatment of disorders characterized by the undesirable release ofhistamine, such as allergy and asthma.

By “stimulated mast cell” is meant a mast cell in an activated statewhich is characterized by, or proximally leads to, degranulation andrelease of mediator from the cell. By “immunologically stimulated mastcell” is meant a mast cell which becomes stimulated by binding ofantigen to IgE on the cell surface. Mast cell immunologic stimulationalso includes experimental immunological stimulation achieved bycontacting mast cells with antibodies to IgE, which results in thecross-linking of attached FcεR receptors on the mast cell.

By “P2-purinoceptor ligand” is meant a compound which binds to aP2-purinoceptor.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of the dose-response relationship of the ATP-modulatedhistamine release from human lung mast cells (HLMC) induced by anti-IgE.ATP at various concentrations was added to cells 15 minutes prior toanti-IgE (3 μg/ml) challenge. Control cells received no ATP.

FIG. 2 is a graph of the potentiation by ATP (10⁻⁴ M) ofanti-IgE-induced histamine release from HLMC. The results are groupedinto preparations in which anti-IgE induced release of histamine wasless than 3% (“low responders”) versus preparations in which anti-IgEinduced release of histamine was equal to or greater than 14% (“highresponders”). Shown are results obtained in 13 out of a total of 20preparations representing extremes of response to ATP.

FIG. 3 is a graph of the comparative modulatory effects of ATP andadenosine on anti-IgE-induced histamine release from HLMC.

FIG. 4 comprises a series of readouts from the high pressure liquidchromatography (HPLC) detection of purine compounds in cell culturemedia containing HLMC cells preincubated with 10⁻⁴ M ATP andsubsequently incubated with or without anti-IgE (3 μg/ml). FIG. 4A:anti-IgE-activated HLMC; FIG. 4B: anti-IgE-activated HLMC+10⁻⁴ M ATP;FIG. 4C, 10⁻⁴ M ATP alone without HLMC (control). The data are theresult of three experiments. The arrow indicates the peak for ATP.

FIG. 5 is a blot of the reverse transcriptase-polymerase chain reaction(RT-PCR) amplification of P2Y₁-, P2Y₂- and P2Y₇-purinoceptor mRNA fromHLMC challenged with either buffer of anti-IgE, followed by extractionof tcRNA.

FIG. 6 is a blot of the RT-PCR amplification of P2X₇/P2Z-purinoceptormRNA from HLMC challenged with either buffer or anti-IgE for two hours.

DETAILED DESCRIPTION OF THE INVENTION

We have shown that ATP can modulate the release of mediators fromstimulated human mast cells. ATP binding to stimulated human mast cellsresults in substantially enhanced mediator release. ATP binding to mastcells presents a target for therapeutic intervention in the treatmentand management of disorders characterized by undesirable mediatorrelease from mast cells.

As demonstrated herein, ATP enhancement of mediator release is notattributable to ectoenzymatic breakdown of ATP to adenosine. Also,adenosine, in contrast to ATP, is observed to exert a bimodal effect onanti-IgE-induced histamine release. At high adenosine concentration,histamine release is significantly inhibited; lower concentrationspotentiated histamine release, though not significantly. Further, inabsolute terms, the ATP enhancement effects were greater than those ofequimolar doses of adenosine.

In addition to ATP, we have found that the pyrimidine nucleotide uraciltriphosphate (UTP), as well as the following ATP analogs, are able tomodulate mediator release from human mast cells: α,βmethylene-ATP(a,βmATP), β,γmethylene-ATP (β,γmATP) and 2methylthio-ATP (2mSATP). Thestructure-function cascade obtained by quantitative analysis of therelative effect of these compounds on histamine release is consistentwith mediation of ATP-induced histamine release by a P2Y-purinoceptor onthe mast cell surface. The finding of ATP modulation of mediator releasefrom mast cells allows, for the first time, a mechanism for regulatingthat mediator release by perturbing ATP binding to its P2-purinoceptoron mast cells. Treatment of mast cell mediator-related disorders may becarried out by administration of molecules, most particularly analogs ofATP, which can competitively bind to P2-purinoceptors on the mast cellsurface and block binding of the authentic receptor ligand ATP.

We have found that the action of ATP in mediating signal transduction inhuman mast cells is entirely different from the action of ATP on ratcells. ATP is able to induce histamine release from unstimulated ratperitoneal mast cells (Keller, Tissue Mast Cells In Immune Reactions, S.Karger, p. 38-39, 1966; Diamant, Int. Arch. Allergy 36:3-21, 1969;Sugiyama, Japan. J. Pharmacol. 21, 209-226, 1971; Cockcroft andGomperts, J. Physiol 296, 229-243, 1979). Surprisingly, we have foundthat ATP alone, in the absence of any stimulatory signal, does not causehistamine release from HLMC. This is in stark contrast to theaforementioned studies wherein ATP alone caused, in a dose-dependentfashion, the direct triggering of histamine release in rat mast cells.Human mast cells which are not first stimulated by cross-linking ofFcεRI surface receptors through antigen or anti-IgE binding, or otherstimulatory signal, do not release mediators upon exposure to ATP.Moreover, it has been suggested that the receptor which mediates theaction of ATP on rat mast cells is the ligand binding channel receptorP2X₇/P2Z, for which the agonist is the tetrabasic form of ATP, ATP⁴⁻(Tatham et al., Euro. J. Pharmacol 147, 13-21, 1988). This ATP⁴⁻receptor is distinct from the P2-purinoceptor which we have foundresponsible for ATP's action on HLMC. ATP⁴⁻ forms complexes with Ca²⁺and Mg²⁺. In our experiments reported herein, negligible amounts ofATP⁴⁻ were present due to the inclusion of both Ca²⁺ and Mg²⁺ atmillimolar concentrations in all assay buffers. Moreover, ATP challengeof HLMC in Ca²⁺-free and Mg²⁺-free media failed to provoke histaminerelease (results not shown).

There is yet further evidence of a different signal transductionmechanism for ATP's action on mediator release from rat versus humanmast cells:

(1) ATP hydrolysis has been viewed as a requirement for rat peritonealmast cell activation (Izushi & Tasaka, Pharmacology 42: 297, 1991). ATPhydrolysis is not required in order to modulate HLMC activation. IntactATP is a modulator of HLMC activation (Example 6).

(2) Rat peritoneal mast cells display a bi-modal response to ATP.Maximum mediator secretion occurs with ATP⁴⁻ at 2 μM, and is depressedby Ca²⁺ and Mg²⁺ (Cockfort & Gomperts, Biochem J. 188: 789, 1980).Stimulated HLMC, in contrast, display a dose-dependent mediator releaseresponse upon ATP binding in the presence of 1 mM each of Ca²⁺ and Mg²⁺(Example 5).

(3) In the presence of millimolar Ca²⁺, ATP⁴⁻ at a concentration above 3μM inhibits mediator release from rat peritoneal mast cells (Bennett etal., J. Physiol. 317: 334, 1981). ATP does not inhibit mediator releasefrom human lung mast cells at any concentration (Example 2).

(4) The ATP analogs α,βmATP and β,γmATP are inactive in inducingmediator release in rat peritoneal mast cells (Id.). These samecompounds are active in enhancing mediator release from HLMC (Example3).

(5) The structure-function cascade of ATP-analog enhancement of mediatorrelease differs in rat peritoneal and human mast cells. For ratperitoneal mast cells, the cascade is 2mSATP≧ATP>>α,βmATP=β,γmATP=0(Tatham et al., Eur. J. Pharmacol 147:13, 1988). The structure-functioncascade for HLMC is ATP>2mSATP≧α,βmATP≧β,γmATP (Example 3).

(6) Rat and human mast cells differ dramatically with respect tosensitivity to UTP. In comparison with ATP, UTP is almost inactive at10⁻⁴M in achieving mediator release from rat peritoneal mast cells(Sugiyama, Japan. J. Pharmacol. 21:209, 1971). But we have found thatUTP is very active in enhancing mediator release from stimulated HLMC(Example 4).

(7) Rat and human mast cells further differ in their response tomagnesium ion. Whereas ImM Mg²⁺ inhibits ATP-induced histamine releasefrom rat cells (Diamant, Int. Arch. Allergy 36:3, 1969), we have foundthat histamine release from HLMC is enhanced by ATP in the presence ofImM Mg²⁺ (Example 2).

(8) Preincubation of HLMC with the putative P2X-purinoceptor antagonistPPADS (Lambert et al., Eur. J. Pharmacol. 217:217-219, 1992) does notaffect ATP modulation of anti-IgE-induced histamine release from theHLMC (Example 9), demonstrating that the ATP receptor on HLMC is amember of the P2Y family, not a member of the P2X family. The receptorwhich mediates the action of ATP on rat mast cells is a member of theP2X family.

According to the present invention, an inhibitor of ATP binding toP2-purinoceptors on human mast cells is utilized to treat humandisorders which are characterized by the undesirable release of mediatorfrom mast cells. By “inhibitor” is meant any agent that is capable of,directly or indirectly, interfering with ATP binding to aP2-purinoceptor which results in a reduction of ATP potentiation ofmediator release from a mast cell. The inhibitor may take the form aP2-purinoceptor antagonist which forms a blockade against ATP binding tothe P2-purinoceptor. Alternatively, the inhibitor may take the form ofan allosteric modifier of the P2-purinoceptor. Such agents act bychanging the conformation of the P2-purinoceptor to reduce receptorbinding affinity for the ligand ATP.

The term “inhibitor” also includes agents which are partial agonists ofATP binding to P2-purinoceptors, and which are consequently competitiveantagonists at the P2-purinoceptor. Those agents which are partialagonists of ATP modulation of human mast cell mediator release areconsidered inhibitory since their binding to the receptor competes withthe authentic ligand, ATP, which has a greater level of activity uponbinding to the P2-purinoceptor than the partial agonist.

Antagonists of P2-purinoceptors include, for example, suramin (Dunn andBlakely, Br. J. Pharmacol. 93:243-245, 1988);pyridzalphosphate-6-azophenyl-2′,4′-disulfonic acid or PPADS (Lambrechtet al., Eur. J. Pharmacol. 217:217-219, 1992);adenosine-3′-phosphate-5′-phosphate or A3P5P;adenosine-3′-phosphate-5′-phosphosulfate or A3P5PS (Boyer et al., Mol.Pharmacol. 50:1323-1329, 1996); and the compound “Reactive Blue 2” whichhas the following structure:

Preferably, the P2-purinoceptor inhibitor is a specific P2Y-purinoceptorinhibitor, most preferably a P2Y₁- or P2Y₂-purinoceptor inhibitor. Thehuman P2Y₁-purinoceptor has been cloned and reported by Schachter etal., Br. J. Pharmacol. 118:167-173, 1996, the entire disclosure of whichis incorporated herein by reference. The human P2Y₂-purinoceptor hasbeen cloned and reported by Parr et al., Proc. Natl. Acad. Sci. USA 91,3275-3279 (1994) the entire disclosure of which is incorporated hereinby reference.

Without wishing to be bound by any theory, it is believed that thereceptor on human mast cells which binds ATP and is thus responsible forATP modulation of mediator release is the same as or similar instructure to the P2Y₁- or P2Y₂-purinoceptor. We have found that purifiedHLMC preparations constitutionally express the P2Y₁- andP2Y₂-purinoceptor (FIG. 5), but not the P2X₇/P2Z-purinoceptor (FIG. 6).The P2X₇/P2Z-purinoceptor is reported to mediate histamine release fromrodent mast cells. We have also found that HLMC do not express theP2Y₇-purinoceptor (FIG. 5).

We have also observed that the structure-function cascade for ATP analogmodulation of histamine release from human mast cells is indicative ofthe structure-function cascade a P2Y-purinoceptor, more particularly theP2Y₁-purinoceptor. With this in mind, the preferred P2-purinoceptorinhibitors for the practice of the present invention areadenosine-2′-phosphate-5′-phosphate or A2P5P, A3P5P, and A3P5PS. Thesecompounds are specific competitive antagonists of the P2Y₁ subtype ofpurinoceptor and do not antagonize other P2-purinoceptors (Boyer et al.,supra). A3P5P and A3P5PS in particular are preferred, as they are devoidof agonist activity at the human P2Y₁ receptor. Partial agonists of P2Y₁include A2P5P and adenosine-2′-phosphate-5′-phosphoribose. Preferably,the P2-purinoceptor inhibitor used in the practice of the presentinvention is a specific inhibitor of ATP binding to theP2Y₁-purinoceptor, which does not bind substantially to otherP2-purinoceptor types, including other P2Y subtypes.

An inhibitor of ATP binding to P2Y-purinoceptors on human mast cells isutilized to treat human disorders which are characterized by theundesirable release of mediator from mast cells. Such disorders includethose conditions which give rise to mast cell stimulation and mediatorrelease. Such conditions include, for example, asthma, allergy,bronchoconstriction and inflammatory lung disease. Mast cells undergoimmunological stimulation by binding of antigen to cell surface IgE.Mast cells, particularly lung mast cells, may also undergo stimulationby nonimmunologic means. For example, mast cells may be stimulated torelease mediator by signals such as contact with cold air, ingestion ofaspirin or aspirin-like drugs, and vigorous exercise.

Pulmonary embolism is associated with massive activation of platelets.Activated platelets release large amounts of ATP. The ATP released fromactivated platelets during acute pulmonary embolism can exacerbatehistamine (and other mediators) release from mast cells and otherinflammatory cells. Exacerbation of histamine release from lung mastcells results in bronchoconstriction. Inhibition of ATP binding toP2-purinoceptors on mast cells is thus particularly useful in thetreatment of bronchoconstriction associated with the acute phase (onset)of pulmonary embolism.

While the principle usefulness of the invention resides in inhibitingATP binding to lung mast cells to counteract bronchoconstriction arisingfrom stimulation of the mast cells and the resulting mediator release,the utility is not limited to modulation of lung mast cell response.Mast cells also populate the skin, nose, eye, gut and skeletal joints.Mast cells of the gut and joints share similar morphology with lung mastcells, and are therefore likely to yield to modulation of mediatorrelease by inhibitors of ATP binding in the same fashion as lung mastcells.

In accordance with the present invention, a compound which inhibits ATPbinding to a P2-purinoceptor may be administered in therapeuticallyeffective amounts in accordance with methods appreciated by thoseskilled in the art. The inhibitor compound is preferably aP2Y-purinoceptor antagonist, more preferably a P2Y₁- orP2Y₂-purinoceptor antagonist. The mode of administration includes anymeans that produces contact of the active ingredient with the site ofaction in the body of a human being, such as in a human body fluid ortissue. These modes of administration include but are not limited tooral, topical, hypodermal, intravenous, intramuscular, inhalational andparenteral methods of administration. In one preferred embodiment of theinvention, the target tissue comprises lung mast cells, and the methodof administration comprises inhalation into or injection into the lung.The P2-purinoceptor antagonist may be administered singly or incombination with other P2-purinoceptor antagonists, or with other activeagents. The antagonists are preferably administered with apharmaceutically acceptable carrier selected on the basis of theselected route of administration and standard pharmaceutical practice.

Methods of administering pharmaceuticals to the lung by inhalation arewell-known to those skilled in the art. The design of suitable inhalerdevices is described, for example in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa., 1985, p. 181-182,incorporated herein by reference.

The dosage of P2-purinoceptor antagonist administered in the practice ofthe therapeutic method of the invention in any particular instance willdepend upon factors such as the pharmacodynamic characteristics of theparticular antagonist; its mode and route of administration; the age,health, and weight of the recipient; the nature and extent of symptoms;the types of concurrent treatment; the frequency of treatment; and theeffect desired. It is contemplated that a daily dosage of aP2-purinoceptor antagonist according to the practice of the presentinvention is in the range of from about 1 μg to about 100 mg per kg ofbody weight, preferably from about 10 μg to about 20 mg per kg of bodyweight, per day. Pharmaceutical compositions may be administered in asingle dosage, divided dosages or in sustained release. Persons ofordinary skill will be able to determine dosage forms and amounts withonly routine experimentation based upon the present disclosure.

The method of therapeutic administration of P2-purinoceptor antagonistincludes administration as a pharmaceutical composition parenterally insterile liquid dosage forms or topically in a carrier. The antagonistmay be formulated into dosage forms according to standard practices inthe field of pharmaceutical preparations. See Gennaro Alphonso, ed.,Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack PublishingCo., Easton, Pa.

For parenteral administration, the P2-purinoceptor antagonist may bemixed with a suitable carrier or diluent such as water, an oil, salinesolution, aqueous dextrose (glucose) and related sugar solutions, or aglycol such as propylene glycol or polyethylene glycol. Solutions forparenteral administration preferably contain a water soluble salt of theP2-purinoceptor antagonist. Stabilizing agents, antioxidizing agents andpreservatives may also be added. Suitable antioxidizing agents includesulfite, ascorbic acid, citric acid and its salts, and sodium EDTA.Suitable preservatives include benzalkonium chloride, methyl- orpropyl-paraben, and chlorbutanol.

According to another aspect of the invention, potential therapeuticcompounds for the treatment of asthma and other disorders characterizedby undesirable mediator release from mast cells, are identified by amast cell assay which relies on mediator release. Test cells comprisingstimulated human mast cells are contacted with a candidate agent whichis an inhibitor of ATP binding to a P2-purinoceptor. The agent ispreferably a compound which is a small molecule suitable for humantherapeutic use. The test cells are then assayed for the release of oneor more mediators. The assay is advantageously carried out as an invitro assay.

The test cells advantageously comprise fresh HLMC. Fresh HLMC may beobtained by a three-day purification protocol which commences withformation of a single cell suspension by enzymatically dispersingfreshly harvested lung tissue, followed by filtration and densityfractionation to obtain an HLMC cell population of greater than 85%purity. The purified HLMC are incubated with the candidate compound,after which ATP is added. The cells are stimulated by addition of aneffective amount of anti-IgE antibody, which simulates cross-linking ofFcεRI receptors by antigen. Cells in a control group are immunologicallystimulated with prior addition of the candidate compound in onesubgroup, and without ATP in another subgroup. The extent of mediatorrelease is determined in all cell groups. The difference between theextent of mediator release by cells treated with ATP and the candidatecompound on the one hand, and cells treated with ATP on the other hand,is a measure of the compound's effectiveness in reducing ATP modulationof mast cells, and the compound's potential usefulness as a therapeuticagent for inhibiting undesirable mediator release.

Preferably, the released mediator which is subject to assay ishistamine. Cell culture supernatant histamine may be measured by anautomated procedure in which histamine is condensed withorthophthaldialdehyde and fluorescence.

According to another aspect of the invention, a screening test forpotential therapeutic agents is provided which relies on assaying of anagent's ability to compete with a P2-purinoceptor ligand for binding toa P2-purinoceptor. The ligand may comprise any compound which is capableof mimicking ATP binding to a P2-purinoceptor. The P2-purinoceptor andligand are preferably a P2Y-purinoceptor and P2Y-purinoceptor ligand,respectively, more preferably a P2Y₁- or P2Y₂-purinoceptor and P2Y₁- andP2Y₂-purinoceptor ligand, respectively.

According to a preferred embodiment, a test compound competes with aP2Y₁-purinoceptor ligand for binding to a reagent comprising aP2Y₁-purinoceptor. A mixture is formed comprising the test compound, aP2y₁-purinoceptor ligand, and a reagent comprising theP2Y₁-purinoceptor. The mixture is then assayed for the ability of thetest compound to inhibit the ligand's binding to the receptor.Inhibition of ligand binding is suggestive of a compound's ability toinhibit mast cell mediator release, and its usefulness as a potentialtherapeutic. A compound proven effective in the ligand binding screenmay then be tested further to establish whether the competitiveinhibition results in P2Y₁-purinoceptor antagonism.

The reagent comprising a P2-purinoceptor in the ligand bindinginhibition assay may be whole cells, cell membranes or fragments of cellmembranes containing the receptor. Preferably, the reagent comprisesfresh HLMC or HLMC membranes. The reagent may also comprise a cell lineexpressing a P2-purinoceptor, such as the cell line HMC-1, derived froma mast cell leukemia patient (Butterfield et al, Leuk. Res. 4:345,1988). The HMC-1 cell line expresses the P2Y₁-purinoceptor.

The P2-purinoceptor ligand in the ligand binding inhibition assayadvantageously comprises a radioactively labeled compound(“radioligand”), and the assay may take the form of a radioligandbinding assay. Radioligand binding assay procedure for biologicalreceptors, and radioligand binding assays for the P2Y₁-purinoceptor inparticular, are known in the art. See for example, Simon et al, Eur. J.Pharmacol., 291, 281-289 (1995) (P2y₁-purinoceptor); Tsukagoshi et al.,J. Pharmacol. Exp. Ther. 273, 1257-1263 (1995) (bradykinin receptor);Belardinelli et al., Circ. Res., 79(6), 1153-1160 (1996) (A_(2A)adenosine receptor). The entire disclosures of Simon et al., Tsukagoshiet al. and Belardinelli et al. are incorporated herein by reference. Fortesting a candidate agent's ability to inhibit ligand binding to theP2Y₁-purinoceptor, the radioligand may advantageously comprise, forexample, [³⁵S]3′-deoxyadenosine 5′-O-(1-thio)triphosphate ([³⁵S]dATPαS)or [³H]uridine 5′-triphosphate ([³H]UTP) (Simon et al., supra). Aliquots(0.5 ml final volume) of freeze-thawed HLMC membrane fraction containingfrom 5-100 μg, preferably 5-10 μg, protein are incubated with drug at aconcentration in the range of 10⁻¹¹-10⁻⁴M and a concentration ofradioligand which is sufficient to saturate the availableP2Y₁-purinoceptors. The effect of the drug on the radioligand binding tothe receptor (specific binding) is determined. Assays are also conductedto identify total and nonspecific binding of the radioligand to thesample. For the specific assay results to have validity, nonspecificbinding of radioligand should not exceed about 30% of radioligand totalbinding to the samples.

The practice of the invention is illustrated by the followingnonlimiting examples.

EXAMPLE 1 Purification of Human Lung Mast Cells

Day 1. Enzymatic Dispersion of Human Lung Tissue. Grossly normal humanlung tissue obtained within minutes of resection is dissected free oftumor, then finely minced and thoroughly washed in divalent cation freeTyrode's buffer. Minced fragments are enzymatically dispersed into asingle cell suspension by two 30 minute incubations at 22 degrees in theenzymes Pronase (2 mg/ml) and chymopapain (0.5 mg/ml), followed by twosimilar incubations in collagenase (1 mg/ml) and elastase Type I (10units/ml). Liberated cells are harvested through Nytex nylon (100 micronpore size) after each digestion and thoroughly washed in Tyrode's bufferto which gelatin (1 g/L), magnesium (1 mM) and deoxyribonuclease (15mg/ml) (TGMD) have been added. Cells (20-100×10⁶, mast cells of 5.6±1.8%purity) are resuspended in culture media consisting of RPMI 1640,L-glutamine (1 mM) and gentamicin (100 μg/ml), and incubated overnightin 100 mm tissue culture plates at 25° C.

Day 2. Elutriation and Dose-response curve. The following morning,non-adherent cells are washed from the plates, then sedimented at 150×gfor 8 minutes. Adherence of cell contaminants and attrition ofcontaminating cells in culture increases mast cell purities to11.4±2.1%. Mast cell recovery is usually complete. Suspensionscontaining 20-100×10⁶ mast cells are subject to counter-currentcentrifugation elutriation (CCE) as follows. The cells are loaded intoan elutriation chamber housed in a Beckman JE21 rotor housed in a J6Bcentrifuge. At a constant rotor speed (1820±5 rpm), buffer (TGMD) flowentering the bottom of the elutriation chamber and flowing in thedirection counter to centrifugal force is increased in pre-definedincrements. Cells are loaded at a buffer flow of 11 ml/minute, then flowincreased to 12, 14, 18, 20, 26 and 30 ml/minute. At each change offlow, 150 ml fractions are collected. The incremental increases inbuffer flow carries cells of ever-increasing diameter out of thechamber. The majority of HLMC, because of their large diameters incomparison to other lung cells, selectively elute in the later fractionsin purities ranging from 20-85%. Cells in each fraction are sedimented,then counted by the Alcian blue technique to determine total cell andmast cell numbers. Fractions most enriched for mast cells are culturedovernight at 37° C., to allow more adherence of contaminatingmacrophages and then further purified over Percoll density gradientfractionations. When time permits on Day 2, a preliminary dose-responsecurve to anti-IgE is performed to access the capacity of cells torespond.

Day 3. Percoll Density Fractionation and Purification. Density gradientfractionation can be performed after CCE on day 2, but the most puremast cell preparations result on Day 3 after overnight culture. HLMCpurification is performed by flotation through discontinuous Percollgradients. Approximately 1-2×10⁷ cells are suspended in 1.0 ml of “100%”Percoll (9 parts Percoll plus 1 part of 10×Hanks' balanced saltsolution, HBSS) and layered at the bottom of a 12×75 mm polystyreneculture tube. Over the cell suspension are layered 0.8 ml aliquots of80%, 70%, 60%, 50% and 40% Percoll solutions, prepared from a stock of100% Percoll. The gradient is then centrifuged at 400×g for 10 minutes;cells at each interface are collected, washed twice in TGMD and counted.Purified HLMC (>85-99% pure) usually float to 60/70%, and/or 70/80%interfaces depending on the properties of mast cells from individuallungs.

EXAMPLE 2 Alternative Method for Purification of Human Lung Mast Cells;Effect of ATP on Histamine Release from Human Lung Mast Cells

A. Buffers

Lung fragments were washed with Tyrode's buffer containing (g/l): NaCl,8.0; KCl, 0.2; NaH₂PO₄, 0.05; and glucose, 1.0. The buffer was titratedto pH 7.2 by the addition of NaHCO₃. Mast cell isolation and elutriationwere performed in a buffer designated “TGMD”, prepared from Tyrode'sbuffer to which the following were added (g/l): gelatin (1.0), magnesium(0.25; 1 mM), and DNase (0.01). The buffer designated “PAGCM” was aPipes-albumin (0.003%) buffer containing (g/l): glucose (1.0),CaCl₂.2H₂O, 0.14 (1 mM); and MgCl₂.6H₂O, 0.2 (1 mM).

B. Human Lung Mast Cells

Mast cells were dispersed from human lung by methods previously reported(Schulman et al., J. Immunol. 29:2662-2667 (1982); Schulman et al., J.Immunol. 131:1936-1941 (1983)). Briefly, lung specimens obtained atthoracotomy for bronchogenic carcinoma were finely minced andextensively washed in divalent cation-free Tyrode's buffer. Fragmentswere briefly incubated in a mixture of pronase (2 mg/ml) and chymopapain(0.5 mg/ml). Freed cells were harvested through Nytex nylon cloth (150microns pore size). Residual fragments were further exposed to a mixtureof collagenase (1 mg/ml) and elastase (10 units/ml). All incubations andwashes were performed at 37° C.; recovered cells were immediately washedthree times in large volumes of TGMD. Mast cell purities in these humanlung cell suspensions ranged from 1-8% as determined by alcian bluestaining (Gilbert et al., Blood 46:279-285 (1975)). Lung mast cells werefurther purified, by counter-current elutriation, using previouslyreported methods (Schulman et al., J. Immunol. 131:1936-1941 (1983)).Mast cells were purified (80->98%) by flotation of enriched elutriationfractions through a discontinuous Percoll gradient (Ishizaka et al., J.Immunol. 130:2357-2362 (1983)). Further mast cell purification wasaccomplished by immunomagnetic negative selection against CD2, CD3, CD4,CD8, CD14, CD16, CD21 and HLADR to ensure against contamination by Tcells, B calls, NK cells, monocytes, and dendritic cells prior to mastcell stimulation using previously described methods (Jaffe et al., Am.J. Respir. Cell. Mol. Biol. 13:665-675 (1995); Jaffe et al., Am. J.Respir. Cell. Mol. Biol. 15:473-481 (1996)).

C. Histamine Release Assay

Mast cells (10-50×10³/tube) were preincubated in either buffer alone orbuffer solutions, each containing ATP for 15 minutes, then challengedwith buffer or anti-IgE at 37° C. in PAGCM. The concentrations ofanti-IgE produce 30-70% of maximal release. Twenty minutes followingactivation, cells were rapidly pelleted and supernatants removed forhistamine analysis. Histamine release was expressed as the net histaminereleased divided by the total histamine content×100%. The total cellularhistamine content was determined following cell lysis with 2% perchloricacid. Spontaneous histamine release was always <2% of cellular histamineand generally <1%. Histamine measurements were performed using theautomated spectrofluorometric method of Technicon (Tarrytown, N.Y.).Variations between replicates were consistently <5%. All assays were runin duplicates.

D. Results

Incubation of purified HLMC with ATP at concentrations ranging from10⁻⁷M-10⁻³M did not directly induce histamine release (n=23). In 20/23preparations in which HLMC responded to anti-IgE stimulation, ATP at10⁻⁴M enhanced histamine release in all (10.9+2.7% histamine release to19.2+2.9% histamine release, p<0.01). In 9 of these 20anti-IgE-responsive preparations (control anti-IgE-induced release of10.1+3.4%, n=9) the dose-dependent effects of ATP were examined from 10⁻⁵M to 10 ⁻³M (FIG. 1). In six of the nine, ATP at 10⁻⁶M was examined.In these six preparations, ATP (10⁻⁶M) had no effects onanti-IgE-induced histamine release. In 9 of 9 experiments, ATP at both10⁻⁵M and 10 ⁻⁴M enhanced histamine release (p<0.05). ATP at 10⁻³Menhanced anti-IgE-induced release in 7/9 experiments and in 2/9,inhibited release. Overall, this enhancement by ATP (10 ⁻³M) to14.0+2.4%, was not statistically significant (p>0.05). In 3/23preparations that failed to respond to anti-IgE alone, preincubationwith ATP (10⁻⁶-10⁻³M) was without effect.

The relationship between the lowest and highest anti-IgE responsivepreparations to the effects of ATP (10⁻⁴M) were contrasted (FIG. 2).Interestingly, ATP enhanced anti-IgE-induced histamine release by ˜8-10%at both extremes. Therefore, in terms of percent enhancement, the ATPeffects were most striking when anti-IgE-induced release was low.Specifically, in experiments with a low (<3%) net anti-IgE-inducedrelease (1.8±0.4%, range 0.5-2.9%, n=6), ATP (10⁻⁴M) enhanced release to13.5±2.7%, (750% enhancement). Anti-IgE-induced histamine release of24.2±4.2% (range 14.0-45.9%, n=7) was enhanced by ATP (10⁻⁴M) to32.9±4.5%, representing only a 35% enhancement.

EXAMPLE 3 Effect of ATP Analogs on Histamine Release from Human LungMast Cells

The procedure of Example 2 was repeated, substituting the following forATP: α,βmethylene-ATP (a,βmATP), β,γmethylene-ATP (β,γmATP) and2methylthio-ATP (2mSATP). In ten experiments, the effect of these ATPanalogues on anti-IgE-induced histamine release were determined.Anti-IgE-induced release of 9.9±3.1% was enhanced by all compounds. In8/10 experiments, ATP itself was the most potent enhancer (17.7±4.1%).In 2/10, 2-mSATP was the most potent, and in 5/10, was the second mostpotent analogue (14.3±3.9%, n=10). The enhancement by the purinenucleotides of histamine release was inversely related to the efficacyof anti-IgE alone in releasing histamine. The structure-function cascadefor the action of the purine nucleotides, wasATP≧2mSATP>α,βmATP>β,γmATP, indicating mediation by a P2Y-purinoceptor(Abbracchio et al., Pharmacol. Ther. 64:445-475 (1994)).

EXAMPLE 4 Effect of UTP on Histamine Release from Human Lung Mast Cells

Because P2Y2 purinoceptors have been shown to be widely expressed inimmune cells, ATP was compared to uracil triphosphate (UTP), thepreferred agonist for this receptor, for effects on anti-IgE-inducedhistamine release. The procedure of Example 2 was repeated, substitutingUTP for ATP. In this group of six experiments, control anti-IgE-inducedhistamine release of 14.9±3.9% was enhanced by ATP (10⁻⁴M) to 23.0±4.7(p<0.05) compared to 19.2±5.0% (p<0.05) in the presence of equimolarUTP. Thus, UTP was less potent than ATP in modulating anti-IgE-inducedhistamine release.

EXAMPLE 5 Effect of Adenosine on Histamine Release from Human Lung MastCells

Since ATP is degraded to adenosine by ectoenzymes (Olsson et al.,Physiol. Rev. 70:761-845 (1990)) and adenosine modulates histaminerelease from rat and human mast cells and basophils (Ott et al., Int.Arch. Allergy. Immunol. 98:50-56 (1982); Church et al., Br. J.Pharmacol. 80:719-726 (1983); Hughes et al., Biochem. Pharmacol.33:3847-3852 (1984); Church et al., Br. J. Pharmacol. 87:233-242 (1986);Peachell et al., Am Rev. Respir. Dis. 138:1143-1151 (1988); Lohse etal., Br. J. Pharmacol. 98:1392-1398 (1989); Post et al., Agents Actions30:30-33 (1990); Peachell et al., J. Pharmacol. Exp. Ther. 256:717-726(1991); Feoktistov et al., J. Clin. Invest. 96:1979-1986 (1995); Ali etal., J. Pharmacol. Exp. Ther. 276:837-845 (1996); Fozard et al., Eur. J.Pharmacol. 298:293-297 (1996)), the effect of adenosine on histaminerelease from HLMC was also determined. The procedure of Example 2 wasrepeated, substituting adenosine for ATP. In six does-responseexperiments (FIG. 3), previous observations (Peters et al., Am. Rev.Respir. Dis. 126:1034-1039 (1982) were confirmed: adenosine alone didnot directly induce histamine release from HLMC, but exerted a bimodalmodulatory effect on anti-IgE-induced histamine release:anti-IgE-induced release of 10.3±3.0% was inhibited by adenosine at 10⁻³M to 5.3±1.9% (p<0.05). At lower concentrations (10⁻⁴M-10⁻⁵M), adenosineenhanced histamine release to 11.2±4.7% and 13.4±5.6%, respectively, butneither effect was statistically significant (n=6). In these sameexperiments, ATP at both 10⁻⁴M and 10⁻⁵M significantly enhancedanti-IgE-induced histamine release.

EXAMPLE 6 Functional EctoATPase Assay of Human Lung Mast Cells

To determine whether the effects of extracellular ATP on purified HLMCmay be mediated in part by degradation to adenosine, the potentialectoenzymatic breakdown of ATP to adenosine was examined by HPLC.Accordingly, HLMC (0.3-1.0×10⁵) in 250 μl PAGCM (n=3) were preincubatedwith 10⁻⁴M ATP for 15 minutes and subsequently incubated with or withoutanti-IgE (3 μg/ml) for an additional 20 minutes. Control preparationswere HLMC without ATP as well as a solution of 10⁻⁴M ATP in PAGCM. Thesupernatants were separated from the cells by centrifugation at14,000-×g for 5 minutes and kept at −20° C. until analyzed by HPLC. Themethod of Stocci et al., Anal. Biochem. 167:181-90 (1987) was used forthe detection of the purine compounds. The HPLC system consisted ofWaters 600E controller, Waters Novapak 4 μm 3.9×150 mm C₁₈ column, and a990 photodiode array detector. The solvent consisted of 0.1 mM KH₂PO₄, 8mM tetrabutylammonium hydrogen sulfate (TAHS) pH 6.0 (buffer A), and 0.1mM KH₂PO₄, 8 mM TAHS pH 6.0 with 30% (v/v) methanol (buffer B). The flowrate was 1 ml/min. with the following gradient program: 100% A to 2.5min., linear gradient to 20% B at 5 min, to 40% B at 10 min., to 100% at13 min., then 100% B to 30 min. 100 μl of supernatant (neat) wasinjected and the separation monitored at 254 nm over the 30 minute runtime. The ATP peak areas were calculated and compared among theconditions. The data are shown in FIGS. 4A-C: 4A, anti-IgE-activatedHLMC; 4B, anti-IgE-activated HLMC+10⁻⁴M ATP; 4C, 10⁻⁴M ATP alone withoutHLMC (control). The data are the result of three experiments.

There was no noticeable decrease in the area under the ATP peak (arrowsat 20 min. in FIG. 4) for anti-IgE activated HLMC in the presence of10⁻⁴M ATP (FIG. 4B) versus the control (10⁻⁴M ATP alone without HLMC)(FIG. 4C). No additional peaks corresponding to ATP metabolites (i.e.,ADP, AMP, adenosine were generated by the anti-IgE-activated HLMC (FIG.4B). The early peak in FIG. 4 is the solvent front artifact and the lowbroad peaks are due to the change in solvent composition.

HLMC thus failed to demonstrate functional ectoATPase activity. Humanlung fragments under identical conditions demonstrated conversion of ATPto adenosine over the 15 minute incubation period (data not shown).

EXAMPLE 7 Effect of ATP Receptor Antagonist on Histamine Release fromHuman Lung Mast Cells

To confirm the effect of a putative P2-purinoceptor antagonist as aninhibitor of mast cell histamine release, the procedure of Example 2 isfollowed, with the following modification. HLMC are incubated for 15minutes with the putative antagonist alone added to the assay at timet=−30 minutes, prior to the addition of buffer or ATP at time t=−15minutes. The effect of the putative antagonist on mast cell activationis determined by comparing the level of histamine release from theanti-IgE-challenged HLMC with and without preincubation of the cellswith receptor antagonist.

EXAMPLE 8 Inhibition of Ligand Binding to P2Y-Purinoceptor

The ability of a candidate pharmacological agent to inhibit ligandbinding to the P2Y₁-purinoceptor on human lung mast cells is determinedas follows. The procedure may be used as a preliminary screen inidentification of possible P2Y₁-purinoceptor antagonists.

A. Preparation of Human Lung Mast Cells Membranes. Fresh human lung mastcells are obtained as in Example 1. A crude membrane fraction is thengenerated according the procedure of Simon et al., Eur. J. Pharmacol.291, 281-289 (1995), the entire disclosure of which is incorporatedherein by reference. Essentially, the harvested HLMC are suspended in abuffer A. Buffer A has the composition: 50 mM Tris/1 mM EDTA/1 mM EGTA,adjusted to pH 7.4 with HCl, and also contains (as protease inhibitors)1 mM benzamidine, 0.1 mM phenylmethylsulphonyl fluoride, 0.01%bacitracin, 0.001% soybean trypsin inhibitor and 40 kallikreininhibition units of aprotinin. The suspended cells are freeze-thawed andfurther disrupted by homogenization with a Ultra-Turrax J-25 homogenizer(2×15 s, setting 5, cooling the suspension for 1 minute between pulses).The membranes are collected by centrifugation at 12000×g, 30 minutes ina microcentrifuge at 4° C. The supernatant is discarded, the membranesare resuspended in buffer A (1 ml) by passing through a 21-gauge sterileneedle and incubated on ice (30 minutes) to chelate endogenous divalentcations, destroy labile endogenous ligands and inactivate traces ofproteases. The membranes are then centrifuged and washed with buffer Atwice. The pellet is resuspended in buffer A to give a proteinconcentration (Bradford, Anal. Biochem. 72, 248 1976) of 0.1-0.2 mg/mland frozen in liquid N₂ before storage at −70° C.

B. Radioligand Binding Assay. A radioligand binding assay is conductedaccording to the procedure of Simon et al., supra. One of the followingP2Y₁ receptor agonist radioligands is used in the binding assay:[³⁵S]3′-deoxyadenosine 5′-O-(1-thio)triphosphate ([³⁵S]dATPαS; 1400Ci/mmol) or [³H]UTP (14 Ci/mmol). Preliminary radioligand binding assaysare conducted to identify total and nonspecific binding of theradioligand to the sample. For the specific radioligand binding assayresults to have validity, nonspecific binding of radioligand should notexceed about 30% of radioligand total binding to the samples.Preliminary radioligand binding assays are also conducted to determinethe concentration of radioligand which is sufficient to saturate all theavailable ligand binding sites on the cells. Specific binding of theradioligand to the receptor is then determined in the absence orpresence of unlabelled candidate drug. Aliquots (0.5 ml final volume) offreeze-thawed membrane fraction containing 5-10 μg protein in buffer Aare incubated with drug at a concentration in the range of 10⁻¹¹-10⁻⁴Mand a saturation concentration of radioligand. The assay is terminatedby rapid filtration through GF/C glass fibre filters (pre-soaked in 20mM sodium pyrophosphate) and the filters are immediately washed with 3×5ml of iced 50 mM Tris/HCl (pH 7.4) on a Millipore vacuum manifold.Filters are dried under an infra-red lamp and their radioactivity isdetermined using Optiphase “HiSafe” II (LKB) scintillant, at a countingefficiency routinely of 95% for ³⁵S and 60% for ³H.

C. Results. The extent of the displacement of the radioactive ligandfrom the receptor by the drug candidate demonstrates the effectivenessof the drug candidate as a competitive inhibitor of ligand binding tothe P2Y₁-purinoceptor. Effective inhibitors may then be tested forantagonism using the HLMC histamine release assay of Example 7.

EXAMPLE 9 Effect of Selective P2X-Purinoceptor Antagonist on HistamineRelease from Human Lung Mast Cells

The procedure of Example 2 was followed, except that HLMC werepreincubated with the selective P2X-purinoceptor antagonistpyridzalphosphate-6-azophenyl-2′,4′-disulfonic acid or PPADS (Lambert etal., Eur. J. Pharmacol. 217:217-219, 1992). In four experiments,anti-IgE-induced control release of 11.2±5.3% was enhanced by ATP(10⁴⁻M) to 15.7±7.1%. Preincubation of HLMC in PPADS (10⁴⁻M) for fifteenminutes prior to addition of ATP (10⁴⁻M), produced no significantmodulation of the ATP effect (16.1±5.9% release).

EXAMPLE 10 P2-Purinoceptor Expression in HLMC

The following experiments demonstrate that ILMC express mRNA for bothP2Y₁- and P2Y₂-purinoceptor, but not for P2X₇/P2Z, the purinoceptorreported to mediate histamine release from rodent mast cells, and notfor P2Y₇, a purinoceptor found in human cell systems.

A. RNA Extraction and PCR

HLMC were challenged with either buffer or anti-IgE for two hours. Totalcellular RNA (tcRNA) was then isolated from the HLMC with purity ≧90%using a modified phenol-chloroform extraction technique adapted fromChomczynski and Sacchi, Anal. Biochem. 162(1):156-159 (1987). Likewise,for positive controls, whole blood was processed by Ficoll-Hypaquegradient centrifugation to obtain peripheral blood mononuclear cells(Jaffe et al., Am. J. Respir. Cell. Mol. Biol. 13:665-675 (1995); Jaffeet al., Am. J. Respir. Cell. Mol. Biol. 15:473-481 (1996)) and cellssimilarly treated for tcRNA. Purified mast cell tcRNA was treated with10 units Heparinase-I (Sigma Co., St. Louis, Mo.) at room temperaturefor 2 hours to neutralize the inhibitory effects of mast cell heparin onRT-PCR reactions (Tsai et al., Am. J. Pathol. 146:335-343 (1995)). cDNAwas synthesized from 1 mg tcRNA using oligo (dT) primers and the murineMoloney leukemia virus reverse transcriptase (Life Technologies, Inc.,Grand Island, N.Y.) at 37° C. for 1 hour in the presence of 20 unitsRNasin with 10 nM each of deoxynucleotide triphosphate (PromegaCorporation, Madison, Wis.). Oligonucleotide probes specific for thefollowing were synthesized: P2Y₁-, P2Y₂-, P2Y₇- andP2X₇/P2Z-purinoceptors; glyceraldehyde-3-phosphate dehydrogenase(GAPDH). Polymerase chain reaction (PCR) was performed using 1 unit TaqDNA polymerase (Life Technologies, Inc., Grand Island, N.Y.) for 30cycles (30 seconds at 94° etc., 30 seconds at 60° etc., 60 seconds at72° etc.) followed by an additional product extension step (72° etc. for5 minutes) using a programmable thermal cycler (GeneAmp 9600, PerkinElmer, Foster City, Calif.). PCR products were separated using agarosegel electrophoresis and visualized by ethidium bromide staining using adigital image analysis system (Gel Doc 1000, Bio-Rad Laboratories,Hercules, Calif.). Amplified PCR products were 370 base pairs for P2Y₁,197 base pairs for P2Y₂, 322 base pairs for P2Y₇, 203 base pairs forP2X₇/P2Z, and 228 base pairs for GAPDH.

B. Results

In 5/5 experiments, HLMC expressed transcripts for P2Y₁-purinoceptor andin 3/3 experiments for P2Y₂-purinoceptor (FIG. 5). P2Y₇-purinoceptor,found in human cell systems, was undetected in 4/5 and faintly expressedin 1/5 (FIG. 5). GAPDH signal was readily detected in all cell samplesat 20 cycles of PCR (FIG. 5). P2X₇/P2Z-purinoceptor expression was notdetected in 5/5 purified HLMC preparations (FIG. 6), although GAPDHsignal was readily detected in all cell samples at 20 cylces of PCR(data not shown).

All references cited with respect to synthetic, preparative andanalytical procedures are incorporated herein by reference.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification, as indication the scope of theinvention.

What is claimed is:
 1. A method for selecting compounds useful forinhibiting histamine release from stimulated human mast cellscomprising: contacting stimulated human mast cells with a candidatecompound which is an inhibitor of ATP binding to a P2Y-purinoceptor; andassaying said cells for release of histamine.
 2. The method according toclaim 1 wherein the compound is an inhibitor of ATP binding to theP2Y₁-purinoceptor or P2Y₂-purinoceptor.
 3. The method according to claim2 wherein the stimulated mast cells comprise immunologically stimulatedmast cells.
 4. The method according to claim 3 wherein theimmunologically stimulated mast cells comprise lung mast cells.
 5. Amethod for determining, in vitro, the effectiveness of an inhibits ofATP binding to a P2Y-purinoceptor as a candidate agent for the treatmentof a human subject for a disorder characterized by undesirable releaseof histamine from stimulated mast cells, the method comprising: forminga mixture comprising the agent, a P2Y-purinoceptor ligand and a reagentcomprising a P2Y-purinoceptor; and assaying the mixture for theinhibition of ligand binding to said receptor by the agent.
 6. Themethod according to claim 5 wherein the reagent comprises theP2Y₁-purinoceptor, and the ligand is a P2Y₁-purinoceptor ligand, or thereagent comprises the P2Y₂-purinoceptor, and the ligand is aP2Y₂-purinoceptor ligand.
 7. The method according to claim 6 wherein thereagent comprises human mast cells.
 8. The method according to claim 7wherein the reagent comprises lung mast cells.
 9. The method accordingto claim 8 wherein the P2Y₁ or P2Y₂-purinoceptor ligand is aradiolabeled ligand.
 10. The method according to claim 9 wherein theradiolabeled ligand is [³⁵S]3′-deoxyadenosine 5′-O-(1-thio)triphosphateor [³H]uridine 5′-triphosphate.
 11. The method according to claim 5wherein the disorder is asthma.